This proposal is a competitive renewal for our grant to determine the physiological role of Fgf23 in vivo. FGF23 regulates phosphate homeostasis and vitamin D metabolism. Changes in serum FGF23 levels in humans result in rickets, osteomalacia, or tumoral calcinosis. We generated Fgf23-/- mice to determine Fgf23 expression patterns and functions. Fgf23-/- mice are severely growth-retarded and displayed hyperphosphatemia, hypervitaminosis D, multiple soft tissue calcifications and ectopic bone nodule formation, osteopenia, osteoidosis, hypogonadism, and a short life span. We crossed this mouse with other transgenic lines to demonstrate significant aspects of Fgf23 physiology, including 1) elevated Fgf23 levels cause rickets in a mouse model of the human disease X-linked hypophosphatemic rickets, 2) Fgf23 regulates serum phosphate levels through sodium-phosphate co-transporter NaPi2a in the kidney, 3) systemic Fgf23 is a regulator of phosphate homeostasis but also has autocrine functions in bone, and 4) vitamin D is a required mediator of Fgf23 signaling. We have expanded our research to explore the interrelationships of Fgf23, PTH and Klotho in more detail. Preliminary work has yielded novel and exciting results, which we will expand upon in our proposed research. First, we will define Fgf23 and Klotho functions that are independent from each other using altered PTH levels. We have obtained exciting data showing that complete ablation of PTH rescues the osteoidosis and mineralization defects in Klotho-/- mice but fails to do so in Fgf23-/- mice. Moreover, we found a marked difference in parathyroid gland size in Fgf23-/-/PTH-/- vs. Klotho-/-/PTH-/- mice despite similar serum biochemistry suggesting that Fgf23 has different effects than Klotho on parathyroid glands. Furthermore, we will examine the outcome of intermittent vs. continuous administration of PTH (1-34) to Fgf23-/- and Klotho-/- mice to detect possible changes in the bone in response to anabolic and catabolic actions of PTH. Moreover, we will investigate whether increased osteopontin (OPN) expression accounts for the bone mineralization defect in Fgf23-/-, Klotho-/-, and Fgf23-/-/PTH-/-. This defect does not occur in Klotho-/-/PTH-/- mice, which exhibit relatively normal OPN levels. We will explore the mechanism responsible for the mineralization defect. Second, we will examine the role of Klotho in specific tissues using our unique Klotho fl/fl mouse. This will allow us to independently examine the effects of Klotho in the kidney and parathyroid glands to assess the relative importance and independent effects of Klotho expression in those tissues. Conditional Klotho fl/fl mice will be challenged either by crossing them with Hyp mice, infusing them with FGF23 protein, or feeding them a low calcium diet. Our proposed research aims to provide novel data regarding the roles of Fgf23, PTH and Klotho in a finer resolution than has been possible so far. This research will provide completely new information about how bone, parathyroid gland, and kidney communicate with each other to regulate mineral ion homeostasis and bone metabolism.